US 2004/0212799 A1 discloses a method for the high spatial resolution imaging of a structure in a sample wherein a substance for marking the structure in the sample is selected from a group of substances which can be changed over by means of a first electromagnetic signal from a first state, in which it has first spectral properties, apart from a region deliberately omitted by the first electromagnetic signal, into a second state, in which it has second spectral properties. Various possibilities are specified for the two spectral properties of the substance. The possibility of the different spectral properties, as also mentioned in the exemplary embodiments described in greater detail in US 2004/0212799 A1, is a first state, in which the substance is fluorescent, and a second state, in which it is non-fluorescent. In order to image the structure in the sample, the fluorescence of the substance is excited by means of a second electromagnetic signal, and the fluorescent light emitted by the substance is detected. In this case, the fluorescent light from the substance can only originate from that region of the sample which is deliberately omitted by the first electromagnetic signal and in which the substance is still in the first state. If said region is for example the zero of an interference pattern of the first electromagnetic signal, it can be made smaller than the diffraction limit that is applicable to the imaging of the sample with light having the wavelength of the first or of the second electromagnetic signal. Among the further possibilities for the two different spectral properties of the substance in the first state and the second state which are discussed in US 2004/0212799 A1, the indication about a different absorption for a second optical signal in the form of a test beam is claimed. However, no further details are found with regard to this variant—corresponding to the preamble of independent patent claim 1—of the known method and the apparatus implicitly disclosed thereby with the features of the preamble of independent patent claim 16.
In order to implement the basic concept known from US 2004/0212799A1 of using an electromagnetic signal to set a spatial distribution of a portion of the substance in a fluorescent state in the case of which the regions in which the substances is present in the fluorescent state are spatially delimited, it is also known for an electromagnetic signal which changes over the substance from a non-fluorescent state into a fluorescent state to be applied to the sample in an intensity such that afterward only individual molecules of the substance are in the fluorescent state, which are imaged separately from one another during a microscopic imaging of the fluorescent light emerging from them. It is thus established that the portions of the fluorescent light from the sample which are detected separately from one another in each case originate from individual molecules whose position can be determined from the centroid of the intensity distribution of the fluorescent light with a higher spatial resolution than the diffraction limit at the wavelength of the fluorescent light. This technique is known not only by the name SPARSE but also by the name STORM or PALM, while the name RESOLFT is used for the technique known from US 2004/0212799 A1.
Methods appertaining to fluorescence microscopy have the known fundamental disadvantage of the bleaching of the fluorescent dyes used. This means that a fluorescent dye can only undergo a finite number of excitation and fluorescence emission cycles, usually fewer than 100 000, before it incurs an undesirable chemical conversion into a state that is permanently no longer fluorescent. This problem also occurs in the case of the RESOLFT and SPARSE techniques described above. An additional factor is that the first electromagnetic signal used to set the spatial distribution of the portion of the substance in the fluorescent state can likewise lead to a burdening and thus possibly earlier bleaching of the substance. Some embodiments of the SPARSE technique therefore provide for bringing the individual molecules of the substance into the fluorescent state only once by means of the first signal and then in that state exciting them to fluorescence until the individual molecule is bleached.
In addition, besides the absolute yield of fluorescent light from a molecule of a fluorescent dye, the quantity of fluorescent light that is obtainable therefrom during a specific period of time is also limited. A molecule of a fluorescent dye can only ever be excited to fluorescence by excitation light when it is in its ground state. Until a next possible excitation it is therefore necessary to wait until the molecule has reverted to its ground state. The fact of whether it emits fluorescent light at all in the event of this reversion to its ground state depends on the ratio of its transition probabilities. Often the emission of fluorescent light occurs in less than 50% of the excitations. The probability with which a fluorescent photon is detected is determined by the respective measurement setup; this value is usually even less than 10%. In order to detect evaluable quantities of fluorescent light, therefore, in many cases long measurement times have to be accepted, in general they are more than 10 μs per pixel or fluorescent molecule. These measurement times accumulate particularly in the case of highly localized portions of the fluorescent dye in the fluorescent state or even individual fluorescent molecules until a complete image of the structure of interest in the sample is created. Long measurement times are not just uneconomic in principle, but regularly pose problems with drifting of various components of the measurement setup used.
As an alternative to marking a structure in a sample with a fluorescent substance, it is known from S. Berciaud et al.: “Photothermal heterodyne imaging of individual metallic nanoparticles: Theory versus experiment” in PHYSICAL REVIEW B 73, 045424 (2006) to mark the structure with gold nanoparticles. In order to visualize the marked structure, a method referred to as photothermal heterodyne imaging (PHI) is described, which is based on an increase in the temperature of the sample by an individual nanoparticle on account of the absorption of an electromagnetic signal by a gold nanoparticle situated in the respective measurement region. The electromagnetic signal absorbed by the gold nanoparticle is converted into heat. This brings about a local temperature increase in the sample at the location of the nanoparticle. In specific terms, a temperature gradient pointing away from the nanoparticle is formed. This temperature gradient affects the phase of a test beam which passes through the sample and which is used in addition to the electromagnetic signal which is absorbed by the gold nanoparticles and thus increases the temperature thereof. For detecting the phase shift on account of a local temperature increase, in order to determine the size and possibly also the position of the temperature increase, the PHI technique involves using a frequency-modulated laser beam for heating the gold particles, which leads to a frequency-modulated phase shift of the test beam, which can easily be detected upon comparison with the non-phase-shifted test beam. It is thereby intended to be possible to detect gold nanoparticles down to a size of 1.4 nm (67 atoms).
Other methods for detecting local temperature increases on account of absorbent gold nanoparticles encompass other forms of far field interference microscopy wherein different portions of a test beam, only one of which portions has passed through the region of the temperature increase, are brought to interference with one another (see D. Boyer et al.: “Photothermal Imaging of Nanometer-Sized Metal Particles Among Scatterers” in Science Vol. 297, pp. 1160-1163 (Aug. 16, 2002).
The effect known as thermal lens can also be used for the microscopic detection of a local temperature increase caused by absorption, wherein here the electromagnetic signal that causes the temperature increase on account of its absorption and the test beam that is phase-shifted owing to the resulting temperature gradient are identical (see E. Tamaki et al.: “Single-Cell Analysis by a Scanning Thermal Lens Microscope with a Microchip: Direct Monitoring of Cytochrome c Distribution during Apoptosis Process” in Analytical Chemistry, Vol. 74, No. 7, pp. 1560-1564 (Apr. 1, 2002)).
There is still a need for a method and an apparatus for the high spatial resolution imaging of a structure in a sample which enable a better spatial resolution of the structure in the sample than the diffraction limit, without accepting the known disadvantages of the use of fluorescent dyes.